Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase

The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123 (1), veramycin A (2), NFAT-133 (3) and benwamycin I (4), which were discovered from Streptomyces species and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of 1−3 was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound 3 was generated. Herein, the PKS assembly logic of 1−4 was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenase nftE(1) and metallo-beta-lactamase (MBL) fold hydrolase nftF(1) were verified as essential genes for the biosynthesis of 1−4. The absence of nftE(1) led to abolishment of 1−4 and accumulation of new products (5−8). Structural elucidation reveals 5−8 as the non-aromatic analogs of 1, suggesting the NftE(1)-catalyzed aromatic core formation. Deletion of nftF(1) resulted in disappearance of 3 and 4 with the compounds 1 and 2 unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF(1) potentially generates the compound 3 through two strategies: catalyze premature chain-offloading as a trans-acting thioesterase or hydrolyze the lactone-bond of compound 1 as an esterase.